Enzymatic degradation of cotton fibers: effect of protein cross-linking and the use of degradation to characterize fibers of plant of different genetic background

ABSTRACT

Specific extraction of the oligomers from cotton fibers can be: achieved by a 24-hr incubation at 37° C. with trypsin, chymotrypsin, proteinase K or pepsin, followed by a second 24-hr incubation at 37° C. with cellulase ( Trichoderma reesei ) or β-glucosidase. Alternatively, samples were first subjected to cellulase or β-glucosidase treatment followed by the protease. The residual material is then treated with 0.5N HCI at 100° C. and the extracts analyzed. Fibers treated with cellulase: followed by protease disintegrated and appeared as a cloudy solution, while the fibers treated with protease followed by cellulase retained their structural identity. This analysis reveals striking differences between cotton fibers from different varieties with respect to their susceptibility to enzymatic degradation. This protocol can be used to identify biochemical characteristics, which can then be correlated with genetic markers for advances in plant breeding.

For the purposes of the United States application based on thisapplication, the present application is a Continuation In Part ofapplication Ser. No. 09/003,679, filed Jan. 7, 1998, which is aContinuation In Part of application Ser. No. 08/516,953, filed on Aug.18, 1995, now issued as U.S. Pat. No. 5,710,047, Jan. 20, 1998; of U.S.Provisional 60/096,162, filed Aug. 11, 1998; and of U.S. ProvisionalPatent 60/106,001, filed Oct. 28, 1998, International Application No.PCT/US99/00368, filed Jan. 7, 1999, and International Application No.PCT/US01/12904, designating the United States, filed 20 Apr. 2001, ofall of which are hereby incorporated by reference into this application.For the purpose of the present International Application and the UnitedStates Application based hereon, the present application is based on andclaims priority from U.S. Provisional Application 60/340,937, filed on10 Jan. 2002 and incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention concerns a method of degrading fibers with specificenzymes and chemical means to characterize degradation products that arerelated to biosynthetic precursors for cell wall biosynthesis. Inaddition, the enzymatic interconversion of soluble precursors fromimmature fibers may also be used. The degradative profiles are differentfor fibers of different varieties that have different fiber properties.This analytical method can be used to assist plant breeding for fiberqualities.

Previously, work in by the present inventor identified glycan oligomersassociated with cotton fibers, cotton fabric, wood and paper.Specifically, glycan oligomers complexed with protein have beenextracted from cotton fibers (Murray, et. al., 2001). Based on thisobservation, specific extraction of the oligomers was attempted.Immature fibers (25 days post anthesis) were subjected to a 24-hrincubation at 37° C. with trypsin, chymotrypsin, proteinase K or pepsin,followed by a second 24-hr incubation at 37° C. with cellulase(Trichoderma reesei) or β-glucosidase. Alternatively, samples were firstsubjected to cellulase or β-glucosidase treatment followed by theprotease. Oligomers were released from fibers by both proteases andcellulase. The residual material was then treated with 0.5N HCl at 100°C. and the extracts analyzed by HPAEC-PAD. Additional oligomers could beextracted from fibers subjected to protease first followed by cellulase,but no oligomers were obtained from the material subjected to thecellulase first followed by the protease. Fibers treated with cellulasefollowed by protease disintegrated and appeared as a cloudy solution,while the fibers treated with protease followed by cellulase retainedtheir structural identity. Chymotrypsin was the most effective proteaseat releasing oligomers and degrading fibers. When mature fibers fromopened bolls were subjected to the same cellulase followed by proteaseprocedure; there was little effect. For this reason, the procedure wasrepeated. At the end of the second cycle, the fibers disintegrated andonly a fine particulate precipitate remained. This precipitate waswashed, and digested in either 0.5N HCl, 2N trifluoroacetic acid or 6NHCl at 100° C. Degradation only occurred in 6N HCl, indicative ofcrystalline cellulose. The resulting monosaccharide composition was atleast 99% glucose. The cellulase from T. reesei was compared tocellulase from T. viride (two sources) and cellulase form Aspergillusniger all of which were not effective in degrading intact fibers.However, the cellulase from T. viride completely degraded the isolatedoligomers. Mature fibers from opened bolls were extracted with coldwater and the extract was removed.

Recent experiments have revealed striking differences between cottonfibers from different varieties, which have differences in fiberquality, with respect to their susceptibility to enzymatic degradationas described here. This degradation protocol by specific enzymaticchallenge can be quantified and rigorously defined. Use of this protocolapplied to fibers of different varieties and different fiber qualitiescan be used to identify biochemical characteristics, which can then becorrelated with genetic markers for advances in plant breeding.

The objective of this invention is to facilitate correlation of fiberstructure at the biochemical level with fiber quality measurements thatcan be used to facilitate breeding for improved fiber quality. Thisinformation can then be used to provide insight to the mechanism(s) bywhich various factors affect fiber quality through environmental and/orgenetic means. Cotton fibers are very complex cellular structures which,at maturity, consist of a waxy cuticle outside a primary cell wallsurrounding a secondary cell wall which, in turn, surrounds a lumencontaining the remnants of the living cell. The secondary cell wall isquantitatively the major component by far. The secondary wall isprimarily made up of cellulose but also contains important constituentsof proteins, lipids, glycoproteins, glycolipids and nucleic acids. Thecellulose content can be an issue of discussion depending on one'sdefinition of cellulose. No doubt the cotton fiber consists of greaterthan 90% β-1,4-glucan, however, the discrepancy may occur to determinethe degree of crystallinity or what proportion of the β-1,4-glucan iscrystalline. There is disagreement among investigators in the fieldabout just what defines cellulose but the one thing they agree on isthat it must have the crystal structure of at least two cellobiosylunits side by side. Therefore cellulose requires more than oneβ-1,4-glucan side by side. The biochemical composition and changes ofconstituents during development of Gossypium hirsutum L. Acala SJ-1 wasreported by Meinert and Delmer (1977). However, chemical compositionalanalysis is only the beginning since it may not provide information onthe organization of constituents. One approach to increase ourunderstanding of cotton fiber wall structure is to degrade fibers in astepwise manner using as gentle a means as possible yet as specific aspossible employing enzymes of known specificity in a stepwise manner.This approach yields more information about the fiber organization thanharsh chemical degradation. A combination of enzymatic and chemicalmethods has been used to analyze cotton fibers.

One approach to the genetic improvement of cotton fiber quality employsthe use of quantitative trait loci (QTLs) associated with agronomic andfiber traits of upland cotton and genetic mapping (Shappley, et. al.,1998, Ulloa and Meredith, 2000, Burr, et. al., 2000). A number of fiberspecific genes have been identified and have been expressed in modelsystems (Burr et al.; http://demeter.bio.bnl.gov). To date, there are nospecific biochemical correlates to fiber quality properties such asstrength, maturity and micronaire. Certainly, cellulose content and thedegree of crystallinity must be factors but their organization has notbeen correlated with fiber measurements. The development of transgeniccotton plants which overexpress specific enzymes or proteins hasprovided some interesting clues which may lead to a better understandingof the fiber wall structure. Haigler and Holaday, 2002, have overexpressed sucrose phosphate synthase (SPS) in transgenic cotton plantswhich increased the available sucrose pool for biosynthetic activities.The result was improved fiber quality including fiber strength and fiberlength. Ruan et al., 2002, report the modulation of fiber quality bymodifying the expression of sucrose synthase. Allen et al., 2002, haveoverexpressed a xyloglucantransferase and a peroxidase in transgeniccotton plants resulting in increased fiber strength and length. Thepresumed mechanism is that the increased xyloglucan transferasefacilitated fiber during the elongation stage of primary cell wallsynthesis. The xyloglucan chains are thought to cross-link the cellulosemicrofibrils. The peroxidase is thought to facilitate ligninbiosynthesis but there is little, if any, lignin in the cotton fibersecondary wall. Both of these cases of the development of transgeniccotton plants resulting in changes in fiber quality suggest that aspecific change, in the case of the xyloglucan transferase, or anincrease early in a biosynthetic pathway may have significant effects onfiber quality as would be expected. It would be of great interest toanalyze the structures of the fibers in both of these cases.

The cotton industry relies a great deal on the measurement of micronaireyet it is not at all clear what biochemical and cellular processescontribute to this measurement and in what manner. It is also not clearjust what biochemical characteristics contribute to fiber strength in aspecific manner. Clearly, the overall fiber properties must be theresult of biochemical characteristics such as the degree ofpolymerization of the β-1,4-glucan chains, the degree of cross-linking,the degree of crystallinity of the cellulose and the linkages betweenthe polysaccharide constituents with the protein and lipid constituents.As is the case with all materials, the properties of the material aredetermined by its molecular structure.

The molecular genetic approach to fiber improvement is to identify genesassociated with fiber development, then attempt to express these genesin model systems and assess their effects on fiber properties. A majorimpediment to understanding the operation of fiber genes has been thatlittle is known about the biochemical pathway of fiber and celluloseassembly. For this reason, the direct application of gene-expressiontechniques to fiber development is likely to produce results that aredifficult to interpret, since there are no biochemical markers to assesseffects of the expressed genes. This is unlike other areas of moleculargenetics where is it possible to monitor various enzymes and biochemicalintermediates that make up a known biochemical pathway. For thesereasons, it is important to characterize cotton fiber wall structure atthe biochemical level.

In previous applications the present inventor described his surprisingdiscovery that it is possible to extract a carbohydrate-containingfraction from properly prepared plant material by a simple cold waterprocess. Essentially, plant tissue is prepared by rapid freezing(preferably by use of liquid nitrogen or solid carbon dioxide) and isthen lyophilized and stored at temperatures below freezing. As disclosedin the above-referenced parent applications carbohydrate-containing cellwall fractions can be easily extracted from the lyophilized tissue bycold aqueous extraction; then, special techniques of High PressureLiquid Chromatography (HPLC) allow resolution of the aqueous extractinto constituent mono and poly-saccharides which can be furtherhydrolyzed to identify the constituent monosaccharides.

The use of high pH anion exchange chromatography with pulsedamperometric detection (HPAEC-PAD) makes possible the unambiguousidentification of cell wall constituents. In HPAEC a salt gradient (suchas a sodium acetate gradient) is applied to a column of special ionexchange resins held at a high pH to sequentially elute various mono andpoly-saccharides. Essentially, the hydroxyl groups of the sugars act asextremely weak acids that become deprotonated at the high pH, binding tothe ion exchange matrix until eluted by the gradient.

While there are a number of vendors of HPAEC materials, the currentinvention has employed products and systems produced by the DionexCorporation of Sunnyvale, Calif. These products and systems areexplained in full in the Dionex Technical Notes, particularly inTechnical Notes 20 and 21, which are hereby incorporated into thisapplication. The carbohydrate fractions isolated from plant cell wallswere analyzed using Dionex CarboPac PA1 and PA-100 columns. Both ofthese columns contain polystyrene/divinylbenzene cross-linked latexmicrobeads (350 nm diameter) with quaternary amine functional groups.The columns were operated under the manufacturer's recommended pressureconditions (4000 psi maximum) in sodium hydroxide eluted with a sodiumacetate elution gradient. When necessary, sugar alcohols were analyzedusing a CarboPac MA1 column that contains porous beads (8.5 μm diameter)of vinylbenzene chloride/divinylbenzene with alkyl quaternary ammoniumfunctional groups

The polysaccharides analyzed in the present invention are appropriatelyreferred to as “glycoconjugates” because they comprise a monosaccharideconjugated to at least one additional monosaccharide (i.e., to form anoligo or polysaccharide) and optionally to a protein or a lipid. As willbe disclosed below at least some of the glycoconjugates comprisepolysaccharides conjugated to a protein moiety. To summarize,glycoconjugates may be polysaccharides, polysaccharides containing aprotein moiety, polysaccharides containing a lipid moiety and/or anycombination of these. In the present application only polysaccharidesand polysaccharides containing a protein moiety have been unambiguouslyidentified. In any case HPAEC characterizes the polysaccharide componentof the glycoconjugate.

SUMMARY OF THE INVENTION

Previously, the inventor identified glycan oligomers associated withcotton fibers, cotton fabric, wood and paper. Specifically, glycanoligomers complexed with protein have been extracted from cotton fibers(Murray, et. al., 2001). Based on this observation, specific extractionof the oligomers was attempted. Immature fibers (25 days post anthesis)were subjected to a 24-hr incubation at 37° C. with trypsin,chymotrypsin, proteinase K or pepsin, followed by a second 24-hrincubation at 37° C. with cellulase (Trichoderma reesei) orβ-glucosidase. Alternatively, samples were first subjected to cellulaseor β-glucosidase treatment followed by the protease. Oligomers werereleased from fibers by both proteases and cellulase. The residualmaterial was then treated with 0.5N HCl at 100° C. and the extractsanalyzed by HPAEC-PAD. Additional oligomers could be extracted fromfibers subjected to protease first followed by cellulase, but nooligomers were obtained from the material subjected to the cellulasefirst followed by the protease. Fibers treated with cellulase followedby protease disintegrated and appeared as a cloudy solution, while thefibers treated with protease followed by cellulase retained theirstructural identity. Chymotrypsin was the most effective protease atreleasing oligomers and degrading fibers. When mature fibers from openedbolls were subjected to the same cellulase followed by proteaseprocedure, there was little effect. For this reason, the procedure wasrepeated. At the end of the second cycle, the fibers disintegrated andonly a fine particulate precipitate remained. This precipitate waswashed, and digested in either 0.5N HCl, 2N trifluoroacetic acid or 6NHCl at 100° C. Degradation only occurred in 6N HCl, indicative ofcrystalline cellulose. The resulting monosaccharide composition was atleast 99% glucose. The cellulase from T. reesei was compared tocellulase from T. viride (two sources) and cellulase form Aspergillusniger all of which were not effective in degrading intact fibers.However, the cellulase from T. viride completely degraded the isolatedoligomers. Mature fibers from opened bolls were extracted with coldwater and the extract was removed.

Recent experiments have revealed striking differences between cottonfibers from different varieties, which have differences in fiberquality, with respect to their susceptibility to enzymatic degradationas described here. This degradation protocol by specific enzymaticchallenge can be quantified and rigorously defined. Use of this protocolapplied to fibers of different varieties and different fiber qualitiescan be used to identify biochemical characteristics, which can then becorrelated with genetic markers for advances in plant breeding.

DESCRIPTION OF THE FIGURES

FIG. 1. shows the effect of cellulase or β-glucosidase on isolatedmultimers; control is the isolated multimers without enzyme treatment.

FIG. 2. The effect of a highly purified cellulase (T. veride) on glycanoligomers from 18 DPA fibers which were precipitated by 80% n-propanol.

FIG. 3. The effect of a highly purified cellulase (T. veride) on glycanoligomers from 18 DPA fibers which were precipitated by 80% n-propanol.

FIG. 4 shows the carbohydrates extracted from the first incubation offibers treated with protease first (chymotrypsin) or cellulase first;PMSF=phenylmethylsulfonyl fluoride, a serine protease inhibitor.

FIG. 5 shows the carbohydrates released from the second incubation ofthe fibers; the cellulase fibers had a first incubation withchymotrypsin and the chymotrypsin fibers had a first incubation withcellulase.

FIG. 6 shows the multimers extracted from the fibers following the twoextraction of FIGS. 4 and 5; CT=chymotrypsin, PMSF=phenylmethylsulfonylfluoride.

FIG. 7 shows the hydrolysis products of the white particle (presumablycellulose) left following the enzymatic digestions of FIGS. 4-6 andsubsequent hydrolysis with 0.1N HCl for 30 min and 2N TrifluoroaceticAcid for 2 hr at 100° C.

FIG. 8. shows a chromatogram of the monosaccharides released from thewhite particles above (FIG. 10) after digestion in 6N HCl for 2 hours at100° C.

FIG. 9 shows carbohydrates released by cellulase without cross-linking(1) or following cross-linking with either 125 mM (2) or 250 mM (3)carbodiimide.

FIG. 10 shows carbohydrates released by chymotrypsin after the cellulasealone treatment (1) or after cross-linking with either 125 mM (2) or 250mM carbodiimide (3) and a cellulase treatment.

FIG. 11 shows carbohydrates released by a second cellulase treatment(following the first cellulase and chymotrypsin treatments) alone (1) orfollowing cross-linking with either 125 mM (2) or 250 mM (3)carbodiimide.

FIG. 12 shows carbohydrates released by a second chymotrypsin treatment(following the first cellulase, chymotrypsin and second cellulasetreatments) alone (1) or following cross-linking with either 125 mM (2)or 250 mM (3) carbodiimide.

FIG. 13 shows the absorbance at 280 nm of carbohydrates released bychymotrypsin indicate the presence of a protein or glycoprotein.

FIG. 14. shows the pattern of oligosaccharides released by variouscellulases from 22 DPA cotton fibers. The peak at about 11 min iscellobiose but there are unresolved peaks on the leading and trailingedge of cellobiose.

FIG. 15. shows the pattern of oligosaccharides released by variouscellulases from 44 DPA cotton fibers. The peak at about 11 min iscellobiose but there are unresolved peaks on the leading and trailingedge of cellobiose.

FIG. 16. shows the residual fibers, if present, (bottom) and theprecipitate from the fourth incubation (second chymotrypsin) forcellulases from T. reesei at pH 4.5, unbuffered, unbuffered+PMSF, T.longibrachiatum at pH 4.5, unbuffered and Humicola insolens at pH 4.5and unbuffered. These were mature, 56 DPA fibers.

FIG. 17. shows a flow chart for analysis of extracts obtained byenzymatic degradation of cotton fibers.

FIG. 18. The fibers remaining in the incubation tubes (lower row) andthe precipitates in the extracts following centrifugation (upper row)for five varieties following the incubation with chymotrypsin which wasthe second incubation. Tubes 1-5 were incubated with the cellulase fromTrichoderma longibrachiatum and tubes 6-10 were incubated with thecellulase from Trichoderma reesei. The varieties by tube were: 1 and 61986 G-2; 2 and 7 Stovepipe; 3 and 8 Tamcot HQ-95; 4 and 9; PaymasterTejas; 5 and 10 Deltapine 90.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor of carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the general principles of the present invention have beendefined herein specifically to provide a method of characterizing plantfibers—especially cotton fibers.

Previously, the inventor identified glycan oligomers associated withcotton fibers, cotton fabric, wood and paper. Specifically glycanoligomers complexed with protein have been extracted from cotton fibers(Murray, et. al., 2001). Based on this observation, specific extractionof the oligomers was attempted. Immature fibers (25 days post anthesis)were subjected to a 24-hr incubation at 37° C. with trypsin,chymotrypsin, proteinase K or pepsin, followed by a second 24-hrincubation at 37° C. with cellulase (T. reesei) or β-glucosidase.Alternatively, samples were first subjected to cellulase orβ-glucosidase treatment followed by the protease. Some of the oligomerswere released from fibers by both proteases and cellulase. The residualmaterial was then treated with 0.5N HCl at 100° C. and the resultingextracts analyzed by high pH anion exchange chromatography with pulsedamperometric detection (HPAEC-PAD). Additional oligomers could beextracted from fibers subjected to protease first followed by cellulase,but no oligomers were obtained from the residual material subjected tothe cellulase first followed by the protease. Fibers treated first withcellulase followed by protease disintegrated, while the fibers treatedfirst with protease followed by cellulase retained their structuralidentity. Chymotrypsin was the most effective protease at releasingoligomers and degrading fibers. When mature fibers from opened bollswere subjected to the same cellulase followed by protease procedure,there was little effect. For this reason, the digestion procedure wasrepeated. At the end of the second cycle, the fibers disintegrated andonly a fine particulate precipitate remained. This precipitate waswashed, and digested sequentially in 0.5N HCl, 2N trifluoroacetic acidand 6N HCl at 100° C. Actual digestion occurred only in 6 N HCl, and theresulting monosaccharides obtained appear a great excess of glucosehowever there are very small quantities of arabinose, galactose, mannoseand several unidentified peaks. The cellulase from T. reesei wascompared to cellulases from T. viride (two sources), Aspergillus niger,T. longibrachiatum and Humicola insolens. Only the cellulases from T.reesei and T. longibrachiatum were effective in degrading intact fibersyet the cellulase from T. viride completely degraded the isolatedoligomers. Mature fibers from opened bolls were extracted with coldwater and the extract was removed.

Enzyme Treatments

Extracted multimers were subjected to incubation with a cellulase(Trichoderma reesei) or a β-glucosidase (almond emulsin). The effect ofthe β-glucosidase appeared to be to increase the heights of the multimerpeaks significantly and to generate one additional small peak with aretention time slightly greater than 20 min. Presumably this is theresult of removing terminal glucose unit(s) that results in a compoundwith an increased detector response. The cellulase gave a very differentresult since it resulted in the near elimination of many peaks and greatreductions in many peak heights with a great increase in the peak heightof the first peak in the series of multimers as shown in FIG. 1.

FIG. 1. shows the effect of cellulase (T. reesei) or β-glucosidase(almond emulsin) on isolated multimers; control is the isolatedmultimers without enzyme treatment. The reader's attention is also drawnto FIG. 2. which shows the effect of a highly purified cellulase (T.veride) on glycan oligomers from 18 DPA fibers which were precipitatedby 80% n-propanol and to FIG. 3. which shows the effect of a highlypurified cellulase (T. veride) on glycan oligomers from 18 DPA fiberswhich were precipitated by 80% n-propanol.

Based on the results of the treatment of the isolated multimers withenzymes, it was decided to attempt to modify the multimers in situ bysubjecting the fibers to a sequential enzyme treatment. The goal was tobe able to specifically remove the multimers by the chemically gentleand specific enzymatic means. If this could be accomplished then onecould make a cogent argument for the multimers as specific components ofthe fiber cell wall. Fibers (25 DPA) were subjected to a 24 hrincubation with trypsin, chymotrypsin, proteinase K or pepsin followedby a second 24 hr incubation at 37° C. with cellulase or β-glucosidase.Alternatively, a duplicate set of samples was subjected to the sameenzymes but in the reverse order. That is the cellulase or β-glucosidasefirst and then the protease second. The final fiber/residual materialwas then subjected to the dilute acid extraction to remove the multimersprior to HPAEC-PAD.

As shown in FIGS. 4-6, material was released by both proteases andcellulase. The multimers extracted from the final residual material(FIG. 6) indicate that multimers could be extracted from the controlfibers or fibers subjected to protease first followed by cellulase, butno multimers were obtained from the material subjected to the cellulasefirst followed by the protease. In that case chymotrypsin was the mosteffective protease just as it was for degradation of the glue. However,the most striking observation was that the fibers treated with cellulasefollowed by protease lost their structural integrity and simply fellapart or were sucked up into the Pasteur pipette when the extract wasremoved.

When mature fibers from opened bolls were subjected to the samecellulase followed by protease procedure, very little happened so theprocedure was repeated a second time. FIG. 7. shows the HPAEC-PADchromatograms of the extracts of the cellulase 1, chymotrypsin 1,cellulase 2, chymotrypsin 2 of mature fibers. Note the peak at 15 minwhich is much greater quantitatively in the chymotrypsin extracts. Atthe end of the second cycle, the fibers completely lost their structuralintegrity and only a precipitate of very small particles remained. Theseparticles were then washed, subjected to digestion either in dilute HCl,in 2N trifluoroacetic acid or in 6N HCl. Actual digestion occurred onlyin 6 N HCl, and the resulting monosaccharides obtained appear to be inexcess of 99% glucose; however there are small quantities of arabinose,galactose, mannose and several unidentified peaks This indicates thatsequential treatment with cellulase followed by protease is an excellentmethod for producing cellulose of extremely high purity. This result isstriking since it provides evidence for significant modification of thefiber walls associated with boll opening and maturity. This means thateven though the cellulosic fiber wall is deposited in daily growthlayers, there is obviously a very significant post-depositionalmodification process that drastically alters the fiber wall properties.

FIG. 8. shows a chromatogram of the monosaccharides released from thewhite particles of FIG. 10 after digestion in 6N HCl for 2 hours at 100°C. Note that the released sugar is essentially exclusively glucose,thereby confirming that the particles are essentially pure cellulose.

Oligomer Protein Complex and Cross-Linking

The probability that cellulose microfibrils of the plant cell wall areembedded in a matrix that “glues” them together has been proposed by anumber of investigators over the years. The nature of such a proteinoligomer complex has been the subject of considerable discussion butthere has been no characterization of such a matrix material. Thepresence of cell wall subunits, in cotton fibers, was proposed by W.Lawrence Balls (Balls, W. Lawrence, 1928, Studies of Quality in Cotton,Macmillan & Co., London.) The present work (see above) on the cell wall“glue” matrix is an extension of work in my laboratory to characterizesoluble oligosaccharides and the sucrosyl oligosaccharides in particularwhich appear to be involved in developmental changes of the cottonfiber.

Mature fibers from opened bolls were extracted with cold water and theextract was removed. Cross-linking was then accomplished usingwater-soluble carbodiimide in unbuffered water at a pH of between 5.0and 5.2. Two concentrations of water-soluble carbodiimide were used, 125mM and 250 mM. The cross-linking reaction was carried out for 2 hr atroom temperature followed by overnight at 4° C. The reaction mixture waswashed from the fibers and the enzymatic digestion then ensued. Thefibers were incubated with cellulase (T. reesei) (1 mg/ml) for 24 hrsfollowed by chymotrypsin (CT) (1 mg/ml) and the incubation sequence wasthen repeated. The results are shown in FIGS. 9-12. In all cases,samples number 1 are the controls; number 2 are the fibers from the 125mM water soluble carbodiimide reaction and number 3 are the fibers from250 mM carbodiimide reaction. Under the reaction conditions thecarbodiimide would be expected to promote amide bond formation betweenamino acids while having negligible effect on ester bond formationbetween carbohydrates.

As explained above, a series of multimers (oligomers) can be extractedfrom developing cotton fibers by both chemical and enzymatic methods.These multimers have retention times of 14 minutes and greater under theanalysis conditions employed. The regular spacing of the peaks isindicative of a series of oligosaccharides varying by a unit monomer insize. These results indicate that the multimers are heteropolymers witha repeating glucan unit extending from a core structure which ispossibly a peptidoglycan. Above it was shown that the structuralintegrity of 25 DPA cotton fibers can be degraded by a sequentialenzymatic treatment with a cellulase followed by a protease whereas thereverse extraction sequence does not result in complete degradation.When fibers from bolls that have opened are subjected to the sameextraction sequence, they do not lose their integrity unless the processis repeated a second time. Following the second protease treatment, thefibers disintegrate into a white particulate precipitate.

Quantitatively the constituents released by enzymatic treatments consistmainly of glucose (Glc) and cellobiose (CB). Carbohydrates released bythe first cellulase treatment are shown in FIG. 9 which demonstratesthat carbodiimide at either concentration dramatically reduced theamount of glucose or cellobiose released by the cellulase treatment. Thepeak at 3.5 min retention time is arabinose. Many more of the peaks inthe 14-20 min range are released by the cellulase from the controlfibers than from the treated fibers. The major peak with a retentiontime of approximately 14.5 min released from the control fibers has adistinctly shorter retention time than the major peak at about 14.65 minreleased from the treated fibers. This is a significant difference andit only is demonstrable in the first cellulase extract.

The carbohydrates released by the first chymotrypsin treatment are shownin FIG. 10. More peaks in the 14-20 min range are released from thecontrol fibers than the treated fibers in addition to the large amountsof glucose and cellobiose released from both treated and controlsfibers. This pattern is consistent for the carbohydrates released by the“second” cellulase treatment (actually a cellulase treatment following achymotrypsin treatment) (FIG. 11) and “second” chymotrypsin treatment(actually a chymotrypsin treatment following a cellulase treatment)(FIG.12).

The carbohydrate peaks released with retention times between 14 and 20minutes also contain a constituent, which absorbs at 280 nm as shown inFIG. 13. The absorbance at 280 nm is usually due to the phenolic aminoacids phenylalanine and tyrosine in proteins although other compoundsmay also absorb at 280 nm. Based on this result along with the materialreleased by the proteases, it is concluded that the carbohydrate peaksin this 14-20 min range are glycoproteins. The observation that linkingwith a carbodiimide renders these carbohydrates more resistant to theprotease release further substantiates the conclusion that they are, infact, glycopeptides. The discovery that the protease digestionsignificantly increases the release of glucose and cellobiose confirmsthat the cellulosic constituents of the wall are cross-linked by aprotease sensitive component (i.e., a protein or glycoprotein).

As detailed above, I have been able to obtain the multimers from a largemolecular complex that is secreted by fibers, in vitro, by a temperaturedependent mechanism. The relative distribution of the multimers can varydepending on the exogenous substrates incubated with the fibers and onthe time of day that the bolls were collected. Under optimal conditionsI have demonstrated the presence of the multimers in an initial solublefraction, a secreted fraction which will not pass through a 0.2 μmfilter, the precipitate of the aqueous extract and the fibersthemselves. The multimers appear to play a structural role in theintegrity of the cotton fiber since experiments to extract the multimersusing specific enzymes resulted in a striking loss of the physicalintegrity of the cotton fibers.

The experiments just described demonstrate the A₂₈₀ profile of thematerial released by the sequential treatment of mature cotton fiberswith cellulase, chymotrypsin, cellulase and then chymotrypsin again.These profiles indicate that the multimers are probably attached toprotein. When the fibers are treated with a water-soluble carbodiimideto form amide bonds between the carboxyl and amino groups of the aminoacid constituents, the fibers become more resistant to enzymaticdegradation. This result shows that bifunctional reagents haveapplications in the textiles and lead to ways to improve the quality(e.g., durability) of cotton fabric. In previous work I have shown thatnormal cotton textiles continually shed water-soluble multimers over thelife of the fabric. This suggests that fabric wear is at least partiallydue to loss of soluble material during washing. Chemical cross-linkingis a way to reduce this loss and, thereby, extend the life of cottonfabrics. Although this test employed carbodiimide any of a large numberof bifunctional reagents known to react with amino groups can be used.These reagents are well known to a person of ordinary skill in the artof protein chemistry. The significant point is that my experiment is thefirst demonstration that protein cross-linking reagents are useful toalter properties of cotton and other plant-based textiles.

Crystalline Cellulose

Hydrolysis experiments on the white particulate material which remainsfollowing the enzymatic digestion of the fibers is consistent with theseparticles being perhaps very highly crystalline cellulose. This resultis consistent with the prediction by Balls (1928) that the fiber wall ismade up of little domino or brick-like structures which are heldtogether and permit the fiber to be flexible. It is probable that thematerial that holds the “bricks” together is the “glue” matrix describedin part here with the multimers attached to a protein backbone. Thisresult is consistent with the fact that plant breeders directly selectfor varieties with different fiber properties including strength. It islikely that a matrix protein is a primary gene product while apolysaccharide, such as cellulose, is the product of a number of enzymeswhich, in turn, are the products of a number of genes. Thus, directselection and manipulation by genetic engineering should be moresuccessful on the matrix protein than on the complex of enzymes neededto synthesize cellulose.

In addition to the T. reesei cellulase, several other cellulases havebeen used to digest cotton fibers. To date, only two cellulases (T.reesei and T. longibrachiatum) have demonstrated the ability to renderthe fibers susceptible to degradation by the proteases. At this time, itis not known if this “activity” could be due to a contaminatingprotease, a contaminating glycosidase or some other factor inherent inthe enzyme preparation. Differences have been noted between the variouscellulases as far as the intermediate oligosaccharides released. All ofthe cellulases are characterized by their sources on the basis ofglucose released from a cellulosic substrate per unit time understandard conditions. These characterizations have all been found to beaccurate. However, the cellulases differ markedly in their intermediatesas is shown in FIGS. 13 and 14.

FIG. 14. shows the pattern of oligosaccharides released by variouscellulases from 22 DPA cotton fibers. The peak at about 11 min iscellobiose but there are unresolved peaks on the leading and trailingedge of cellobiose. (The peak at the leading edge of cellobiose has alsobeen seen in this laboratory in biosynthetic experiments. The peaks fromboth experiments co-chromatograph.)

FIG. 15. shows the pattern of oligosaccharides released by variouscellulases from 44 DPA cotton fibers. The peak at about 11 min iscellobiose but there are unresolved peaks on the leading and trailingedge of cellobiose. FIG. 16. shows the residual fibers, if present,(bottom) and the precipitate from the fourth incubation (secondchymotrypsin) for cellulases from T. reesei at pH 4.5, unbuffered,unbuffered+PMSF, T. longibrachiatum at pH 4.5, unbuffered and Humicolainsolens at pH 4.5 and unbuffered. These were mature, 56 DPA fibers.

To date the following cellulases have been compared using 50 DPA fibers:

-   -   1. Aspergillis niger (Sigma):    -   2. Trichoderma veridie (Sigma),    -   3. Trichoderma veridie (Megazyme), Humicola isolens (Fluka),    -   5. Trichoderma reesei (Sigma) Trichoderma veridie (Fluka),    -   7. Trichoderma reesei (Fluka), Trichoderma longibrachiatum        (Fluka).

Of these cellulases tested, only Trichoderma reesei (Sigma) andTrichoderma longibrachiatum (Fluka) rendered the fibers susceptible toprotease degradation. The fact that different cellulases releaseddifferent oligosaccharides and that the same cellulase releaseddifferent oligosaccharides under different pH conditions may not beterribly surprising and, in fact, may be useful to the characterizationof cotton fibers from different varieties. The fact that the T. reeseiand T. longibrachiatum cellulases are the only ones which rendered thefibers susceptible to the protease may be due to the presence ofswollenin (Saloheimo, et. al., 2002 and Swanson, et. al., 2002) which isa protein which disrupts cellulose fibril structure but does not cleaveglycosidic bonds.

In all cases, an attempt was made to use equivalent amounts of cellulasein all incubations. This was done by comparing the specific activity ofeach cellulase as stated by the supplier on the label of the bottle. Inall cases, the activity is based on glucose or reducing sugar releasedfrom a known substrate of SigmaCel 20 or carboxymethylcellulose. Basedon the results obtained, it is obvious that it is not valid to comparethe different cellulases based on the glucose or reducing sugarreleased. Clearly, different intermediate products are released alongthe pathway between the macromolecular substrate and glucose.

There is a large body of literature dealing with the action ofcellulases on cotton fibers as well as other substrates. However, thatwork has been done on cotton fibers that have been subjected toprocessing treatments since the goal of most of that work is tofacilitate fabric finishing and other applications in the textileindustry. The majority of this recent work has been done using theendocellulase and cellobiohydrolase from Trichoderma reesei (Lee, et.al., 2000, Pere, et. al., 2001, Väljamäe, et. al., 2001) but the use ofrecombinant enzymes from Humicola insolens involved twocellobiohydrolases and one endocellulase (Boisset, et. al., 2001). Inall of these studies the activities were assayed by measuring the endproducts, cellobiose or glucose and the intermediate products were notinvestigated. The possibility that these cellulases might also functionas glycosyl transferases and may thus actually synthesize intermediatescan not be overlooked in the light of reports of synthesis ofβ-lactosides by Trichoderma reesei cellulase (Totani, et. al., 2001).

Comparison of Enzymatic Degradation of Different Varieties

Fibers of different varieties have been subjected to the degradationprotocol shown in FIG. 16. The results of the analyses can be comparedto the genetic background of the varieties to determine if correlationscan be made. The differences are clearly striking. It will be importantto compare varieties which are more closely related genetically todetermine just how little a difference maybe detected. One would assumethat differences which are specific to the cell wall would be detectablein very closely related varieties while differences which are notspecific to the cell wall, may not be as striking. To date, overnightincubations have been used due to the convenience and not knowing ifshorter incubation periods may work just as well. To date allincubations have been at 37° C. with toluene layered over the mixture toinhibit microbial growth. However, some investigators have assayedcellulases at 45° C. It may also turn out that one temperature may beoptimal for a cellulase and another may be optimal for a protease.

A flow chart for the analytical procedure is shown in FIG. 17 for oneenzyme incubation. The area in the box indicated by the dotted line isthen be repeated following each enzyme incubation. The constituentsidentified and quantified by HPAEC-PAD and entered into a balance sheetfor comparison between varieties. Differences are detected betweenvarieties. However, varieties compared must come from variety trials inthe same field to rule out differences due environmental variation.

FIG. 18 shows the fibers remaining in the incubation tubes (lower row)and the precipitates in the extracts following centrifugation (upperrow) for five varieties following the incubation with chymotrypsin whichwas the second incubation. Tubes 1-5 were incubated with the cellulasefrom Trichoderma longibrachiatum and tubes 6-10 were incubated with thecellulase from Trichoderma reesei. The varieties by tube were: 1 and 61986 G-2; 2 and 7 Stovepipe; 3 and 8 Tamcot HQ-95; 4 and 9; PaymasterTejas; 5 and 10 Deltapine 90.

The dry mass of the precipitates released by each enzyme incubation canbe determined. The oligosaccharides released can be quantified fromchromatograms showing the sugars released by the enzyme treatment. Thedifferences are clear but they are easily summarized in a more strikingmanner in the photograph of FIG. 18.

The rationale for the enzyme degradation experiments, as stated earlier,was to attempt to remove the glycan oligomer complex specifically withcellulases and proteases to determine it played a structural role. Thefact that the fibers fall apart was a surprise and just adds furtherevidence for a structural role. The rationale for the comparativedegradation experiments on different varieties of cotton is based on thedifferences in fiber properties. Clearly, fiber properties such asstrength, micronaire and maturity are each the composite representationof a number of characteristics at the biochemical level. Thesecharacteristics may be the result of the total amount of cellulose, themanner in which the cellulose is complexed with protein or other wallconstituents. The possible combinations of linkages, includingβ-1,3-glucans, β-1,4-glucans, arabinogalactans in cotton fiber walls(Bucheli, et. al., 1985, 1987, Buchala and Meier, 1981), all playcritical roles in contributing to fiber strength, micronaire andmaturity. When these factors are considered in the light of one reportthat the cotton fiber has acquired about 60% of its strength before themajor contribution of secondary cell wall synthesis (Lewis and Benedict,1994) the concept of the complexity of the cotton fiber wall is trulyamazing. The number of gene products which contribute to the fiber wallclearly must be very large and encompasses precursors of proteins,lipids, glycoproteins, glycolipids as well as polysaccharides. The roleof cellulases or β-glucanases in cellulose biosynthesis and cell wallbiosynthesis is not understood but they are required and their role hasrecently been reviewed (Mølhøj, et. al., 2002) By systematic challengesto the biochemical integrity of the fiber wall and the resultantspecific degradation, it is possible to characterize differences betweenfibers of differing quality at the biochemical level. Further, if thisis done to different varieties grown under identical conditions, to ruleout environmental differences, then this approach will facilitate thesearch for biochemical differences between different

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust-described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

REFERENCES

-   Allen, R. D., Y. Kasukabe, I. Ihara, and Y. Maekawa, 2002, Cotton    plants with improved fiber characteristics and method for producing    cotton fibers from these cotton plants, U.S. Patent Application No.    U.S. 2002/0049999 A1.-   Boisset, C., C. Pétrequin, H. Chanzy, B. Henrissat and M. Schülein,    2001, Optimized Mixtures of Recombinant Humicola insolens Cellulases    for the Biodegradation of Crystalline Cellulose, BIOTECHNOLOGY AND    BIOENGINEERING 72: 339-345.-   Buchala, A. J. and H. Meier, 1981, An Arabinogalactan From The    Fibres Of Cotton (Gossypium arboreum L.), Carbohydrate Research    89:137-143.-   Bucheli, P., A. J. Buchala and H. Meier, 1987, Aurolysis in viotro    of Cotton (Gossypium hirsutum) Fibre Cell Walls, Physiol. Plantarum    70:633-638.-   Bucheli, P., M. Dürr, A. J. Buchala and H. Meier, 1985, β-Glucanases    in Developing Cotton (Gossypium hirsutum L.) Fibres, Planta    166:530-536.-   Haigler, C. H. and A. S. Holaday, 2002, Transgenic cotton plants    with altered fiber characteristics transformed with a sucrose    phosphate synthase nucleic acid, U.S. Pat. No. 6,472,588 B1.-   Liu, S., S. Saha, D. Stelly, B. Burr, and R. G. Cantrell. 2000.    Chromosomal assignment of microsatellite loci in cotton. J. Heredity    91:326-32.-   Lewis, H. L. and C. R. Benedict, 1994, Biochemistry of Cotton,    Biochemistry of Cotton Workshop (Jividen, G. M. and C. R. Benedict,    eds.) Galveston, Tex., pp 212-123.-   Lee, I., B. R. Evans, J. Woodward, 2000, The mechanism of cellulase    action on cotton fibers:evidence from atomic force microscopy,    Ultramicroscopy 82:213-221.-   Meinert, M. C. and Delmer, D. P., 1977, Changes in Biochemical    Composition of the Cell Wall of the Cotton Fiber During Development,    Plant Physiol. 59, 1088-1097.-   Mølhøj, M, Pagant, S and H. Höfte, 2002, Towards Understanding the    Role of Membrane-bound Endo-β-1,4-glucanases in Cellulose    Biosynthesis., Plant Cell Physiol. 43(12): 1399-1406.-   Murray, A. K., R. L. Nichols, and G. F. Sassenrath-Cole, 2001, Cell    Wall Biosynthesis: Glycan Containing Oligomers in Developing Cotton    Fibers, Cotton Fabric, Wood and Paper Phytochemistry, 57(6):975-986.-   Peng, L., F. Xiang, E. Roberts, Y. Kawagoe, L. C. Greve, K. Kreuz    and D. P. Delmer, 2001, The Experimental Herbicide CGA 325'615    Inhibits Synthesis of Cryastalline Cellulose and Causes Accumulation    of Non-Crystaline β-1,4-glucan Associated with CesA Protein, Plant    Physiol. 126:981-992.-   Pere, J., A. Puolakka, P. Nousiainen and J. Buchert, 2001, Action of    purified Trichoderma reesei cellulases on cotton fibers and yarn,    Journal of Biotechnology 89:247-255.-   Ruan, Y-L., R. Furbank, D. Llewellyn, 2002, Modification of sucrose    synthase gene expression in plant tissue and uses therefore, U.S.    Patent Application No. U.S. 2002/0116736 A1.-   Saloheimo, M., M. Paloheimo, S. Hakola, J. Pere, B. Swanson, E.    Nyyssönen, A. Bhatia, M. Ward and M. Penttilä, 2002, Swollenin, a    Trichoderma reesei protein with sequence similarity to the plant    expansins, exhibits disruption activity on cellulosic materials,    Eur. J. Biochem. 269, 4202-4211.-   Swanson, B. A., M. Ward, M. Penttilä, J. Pere, and M. Saloheimo,    2002, Microbial swollenin protein, DNA sequences encoding such    swollenins and a method of producing such swollenins, U.S. Pat. No.    6,458,928 B1.-   Totani, K., N. Yasutake, H. Ohi, T. Murata, and T. Usui, 2001,    Enzymatic Synthesis of Aliphatic b-Lactosides as Mimic Units of    Glycosphingolipids by Use of Trichoderma reesei Cellulase, Archives    of Biochemistry and Biophysics 385:70-77.-   Shappley, Z. W., J. N. Jenkins, J. Zhu, and J. C. McCarty,    Jr., 1998. Cotton Improvement, Quantitative Trait Loci Associated    with Agronomic and Fiber Traits of Upland Cotton, J. Cotton Science    2:153-163.-   Ulloa, M. and W. R. Meredith, Jr., 2000. Breeding & Genetics,    Genetic Linkage Map and QTL Analysis of Agronomic and Fiber Quality    Traits in an Intraspecific Population, J. Cotton Science 4:161-170.-   Väljamäe, P., G. Pettersson and G. Johansson, 2001. Mechanism of    substrate inhibition in cellulose synergistic degradation, Eur. J.    Biochem. 268, 4520-4526.

1. A method of improving fabric properties comprising treating cottonfibers with a chemical reagent which forms covalent bonds with aminogroups present within the fibers.
 2. The method of claim 1, wherein thechemical reagent is a carbodiimide.
 3. The method of claim 1, whereinthe chemical reagent forms amide bonds.
 4. A method of enzymaticallydegrading cotton fibers to yield essentially pure cellulose comprisingthe steps of sequentially treating the fibers first with cellulase andthen with protease.
 5. A method of characterizing cotton fiber cellwalls comprising the steps of specific enzyme degradation in sequentialsteps utilizing cellulases and proteases.
 6. The method of claim 5,wherein the cellulases are utilized at different pH's to accentuatedifferences between cotton fibers of different varieties.
 7. The methodof claim 5, wherein different types of proteases are utilized.
 8. Themethod of claim 7, wherein the different types of proteases are utilizedsequentially.
 9. The method of claim 5, wherein different types ofcellulases are utilized.
 10. The method of claim 9, wherein differenttypes of cellulases are utilized sequentially.
 11. The methods of claim5-10, further comprising the step of utilizing the characterization ofcotton fibers according to the methods to develop biochemical markersfor fibers of different cotton varieties.
 12. The method of claim 11,wherein the biochemical markers are used in plant breeding to improvefiber quality.
 13. The method of claim 11, wherein the biochemicalmarkers are used as a means to distinguish varieties of cotton.